Immunology


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0 Q&A 4989 Views May 20, 2021

The recombinant receptor-binding domain (RBD) of the viral spike protein from SARS-CoV-1 and 2 are reliable antigens for detecting viral-specific antibodies in humans. We and others have shown that the levels of RBD-binding antibodies and SARS-CoV-2 neutralizing antibodies in patients are correlated. Here, we report the expression and purification of properly folded RBD proteins from SARS and common-cold HCoVs in mammalian cells. RBD proteins were produced with cleavable tags for affinity purification from the cell culture medium and to support multiple immunoassay platforms and drug discovery efforts.


Graphic abstract:



High-Yield Production of Viral Spike RBDs for Diagnostics and Drug Discovery


0 Q&A 3611 Views Jun 20, 2020
Human leukocyte antigen class I (HLA-I) molecules are a group of structurally-related cell surface proteins with a high degree of variability within the population. While only up to six variants are expressed in an individual person, the whole population contains thousands of different variants. The ability to distinguish specific variants is important in the clinic to determine compatibility during organ and bone marrow transplantation and in the laboratory to study the biological properties of individual variants. Solid phase bead arrays contain purified, individually identifiable HLA-I molecules that can be used to determine antibody specificity for individual HLA-I proteins. This method is high-throughput, highly specific, and allows for simultaneous screening of antibodies against multiple HLA-I allotypes. The beads are particularly useful for screening patient sera for the presence of donor-specific antibodies against individual HLA-I variants (which can arise during pregnancy, blood transfusion, or organ transplantation). Alternate approaches, such as the use of individual HLA-I-expressing cell lines, are more time consuming, and such cell lines are difficult to procure and standardize. The HLA-I beads are also useful to study HLA-I specificity and selectivity for other receptors and binding partners.
0 Q&A 7575 Views Jun 20, 2019
Non-infectious virus-like particles (VLPs) containing dengue virus (DENV) pre-membrane (prM) and envelope (E) proteins have been demonstrated to be highly immunogenic and can be used as a potential vaccine candidate as well as a tool for serodiagnostic assays. Successful application of VLPs requires abundant, and high-purity production methods. Here, we describe a robust protocol for producing DENV VLPs from transiently-transformed or stable COS-1 cells and further provide an easily adaptable antigen purification method by sucrose gradient centrifugation.
0 Q&A 5491 Views Feb 20, 2019
Membrane proteins such as cytokine receptors and G protein-coupled receptors can be drug targets. Recently, we have generated specific monoclonal antibodies (mAbs) against the mouse IL-9 receptor (IL-9R) and found that IL-9R on memory B cells have critical roles in T-dependent immune response. So far, most antibodies against cell surface proteins have been generated by immunization of animals with recombinant proteins produced in Escherichia coli (E. coli) or peptides derived from the protein. However, such antibodies often fail to recognize native proteins on cell surfaces because these antigens lack posttranslational modification and natural protein conformations. To circumvent such problems, we have developed a mouse immunization method, the DNA-immunization utilizing hyaluronidase and E. coli GroEL. Herein, we report an application of the original mouse immunization method in rats to generate anti-mouse IL-9R mAbs which could react with the native form of mouse IL-9R on cell surfaces. Thus, we suggest that the DNA-immunization method is feasible for generating monoclonal antibodies against cell surface proteins in rats.
0 Q&A 7754 Views Oct 5, 2015
Kinetic analysis of antibodies is one of the important studies for characterization of antibodies and screening of ligands. In our recent study (Ingale et al., 2014), we compared the antigenic profiles and binding characteristics of four HIV-1 envelope glycoprotein (Env) core immunogens using multiple monoclonal antibodies by Bio-Layer Light Interferometry (BLI). This technology enables real-time analysis of interactions on the surface of a fiber optic biosensor by accurately measuring kinetic constants such as Ka, Kd, and KD in a 96-well format.
1 Q&A 10668 Views Dec 20, 2014
The fluorescence-linked antigen quantification (FLAQ) assay allows a fast quantification of HIV-1 p24Gag antigen. Viral supernatant are lysed and incubated with polystyrene microspheres coated with polyclonal antibodies against HIV-1 p24Gag and detector antibodies conjugated to fluorochromes (Figure 1). After washes, the fluorescence of microspheres is measured by flow cytometry and reflects the abundance of the antigen in the lysate. The speed, simplicity, and wide dynamic range of the FLAQ assay are optimum for many applications performed in HIV-1 research laboratories.
0 Q&A 15448 Views Nov 5, 2014
Surface Plasmon Resonance (SPR) is widely used to generate kinetic and affinity information on specific interactions between biomolecules. This technique is label-free and monitors the binding event in real-time. It is generally used for characterization of monoclonal antibody - antigen interactions. This protocol describes specifically the use of SPR with a Biacore T100 instrument to measure the affinity of crude hybridoma samples to a protein. For that purpose an anti-IgG antibody was firstly covalently immobilized onto a CM5 chip by amide coupling (Canziani et al., 2004; Schraml and Biehl, 2012). Then the antibodies from hybridoma supernatants were captured non-covalently onto the surface via their Fc region providing an optimal analyte-binding orientation. Finally, the resulting complex was stabilized by crosslinking with EDC/NHS to avoid baseline drift during measurement and regeneration (Pope et al., 2009). Then the interaction with the protein was monitored at several concentrations and its affinity towards the immobilized antibodies was determined with the corresponding KD obtained from classical kinetics analysis. This set-up avoids the avidity effects of the bivalent antibodies, allows the use of non-purified analytes with unknown concentrations and the specific capture of the antibodies in a similar stable covalent-orientated manner.
0 Q&A 8806 Views Apr 5, 2014
α2β1-integrin clustering experiment can be used to trigger internalization of α2β1-integrin. When clustering is performed with sequential administration of primary and fluorescent secondary antibodies, the entry kinetics of integrin can be followed into the cell. The idea is first to allow binding of primary antibodies (recognizing the extracellular epitope) to the α2β1-integrins and then to cluster the α2β1-integrin-bound primary antibodies together by the means of the secondary antibody. Binding is done on ice so that the α2β1-integrins will not internalize before both sets of antibodies are bound. Clustering is known to trigger α2β1-integrin internalization efficiently from the cell surface to the cytoplasm. In this protocol we used antibody-induced clustering of α2β1-integrin in order to quantitate the amount of internalized α2β1-integrins in comparison to cell surface-associated α2β1-integrin.
0 Q&A 9472 Views Feb 20, 2014
This protocol will result in the accurate qualitative measurement of anti-Chikungunya virus antibody (Ab) from infected mouse tissue or serum. This assay was developed by Dr. Caitlin Briggs, Arbovax, Inc. Chikungunya is a BL3 agent and should be handled in a biosafety level 3 laboratory under BL3 conditions. This protocol was used in the publication “Chikungunya virus host range E2 transmembrane deletion mutants induce protective immunity against challenge in C57BL/6J mice” (Piper et al., 2013).
0 Q&A 12565 Views Dec 20, 2013
Molecular interaction between monoclonal antibodies (MAbs) and their recognized antigen is a fundamental event leading to the neutralization activity. Estimation of their binding affinity gives beneficial information to characterize the MAbs and to develop more effective MAbs. Surface plasmon resonance (SPR) analysis is a powerful tool to analyze the molecular interaction, enabling rapid and repetitive estimation with relatively small amount of sample. Here we describe a general protocol about SPR analysis on the interaction between viral antigen and human MAb (HuMAb) IgG. Anti-human Fcγ is first covalently crosslinked on the sensor chip by amine coupling, and then HuMAb of interest is immobilized via anti-Fcγ MAb IgG interaction as ligand. Antigen injected on the sensor chip causes the SPR change in time course as the result of association and dissociation. By analyzing the kinetics, association rate, dissociation rate, and dissociation constant are obtained.



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